The present invention is directed to a self-assembled nano-array molecular sieve for the separation of molecules.
This invention relates in general to self-assembled nanometer-scale arrays used as molecular sieves in the separation of molecules by differential transport through the array, and in particular to self-assembled carbon nanotube arrays used as electrophoretic sieves for DNA sequencing and separation of biological molecules comprising a self-assembled carbon nanotube array arranged on a substrate and an electromagnetic field generator for applying a potential across the array sieve producing a characteristic mobility in the molecules.
Electrophoresis is the predominant technique for separating DNA fragments obtained from restriction maps of complete genomes (millions of base pairs long) and for large-scale sequencing projects, like the Human Genome Initiative. Electrophoresis has also become an essential tool for clinical chemistry applications. In conventional electrophoretic sieves, electrophoretic separation occurs by differential transport of polyelectrolytes, such as, DNA molecules and proteins, through a medium or device in the presence of an electric field. The medium acts as a sieve, producing a size-dependent mobility in the molecules. In slab gel electrophoresis, the sieving medium is provided by a slab gel of agarose or polyacrylamide polymers, which contain nanometer-size pores. For example, an agarose gel is made by placing agarose into solution with a suitable solvent and the pore size depends on the concentration of agarose in solution according to the equation:
xcex1≈89xc2x7Axe2x88x922/3 nmxe2x80x83xe2x80x83(1) 
where a is the pore size and A is the concentration of the agarose in (g/mL). Pore size for an agarose gel with Axcx9c0.1 to 1.0 g/mL is in the range of 100 to 500 nm.
Polyacrylamide polymers are produced through a polymerization reaction of acrylamide and methylenebisacrylamide. By controlling the conditions of the reaction, such as acrylamide concentration and the degree of cross-linking, the pore structures thus formed can be reproducibly controlled and can have pore sizes as small as a few nanometers. Because of the small pore size, polyacrylamide gel electrophoresis (PAGE) is the method of choice for separating small DNA strands ( less than 1000 bases) for DNA sequencing or genetic mutation studies, such as cancer detection or toxicology.
Despite the wide-range of pore sizes available, and the well-documented reproducibility of the conventional gel electrophoresis techniques, severe throughput limitations, size limitations and the need for cleaner, hardier and more user-friendly technologies have led a number of researchers to look for ways to improve the automation and rapidity of electrophoretic techniques. For example, sequencing speed is limited in gel electrophoresis because the electric fields used to push the molecules through the sieve must be kept low to avoid Joule heating of the gel, which could cause degradation of the gel material. In addition, gels are not very durable, requiring constant replacement which results in the further requirement for extra-plumbing and reservoirs to allow facile replacement of the gel adding to both the size and complexity of the gel-electrophoresis equipment.
One recent advance has been capillary electrophoresis (CE). In capillary electrophoresis, a small diameter capillary acts as the sieve for the molecules. CE has gained widespread popularity because the small-scale CE sieve structures allow for the facile dissipation of excess heat, which in turn allows for the use of higher electric fields resulting in a reduction in sequencing time. However, CE systems still contain gels and would thus not be appropriate for extended periods of use, because of the added complexity of storing replaceable gels and injecting them periodically in the micro-channel structure.
Hybridization techniques have offered some promise for rapid separation. However, technical issues in data collection, such as low signal-to-noise ratios and analysis, such as computationally intensive combinatorial analysis have prevented hybridization techniques from becoming the standard in DNA analysis.
More recently, researchers at Princeton have introduced micro-fabricated arrays as artificial sieving structures to replace polymer-based sieves. These artificially fabricated arrays have several advantages compared to both the polymer gels and the capillaries including: 1) the possibility of using ultra-high fields enabling higher speed separation and real-time monitoring of DNA samples; 2) the use of a non-viscous medium leading to higher durability and which in turn could possibly lead to the development of permanent sieves; 3) the flexibility and controllability of the configuration of the sieving structure would allow for analysis over a broad range of molecular sizes, from simple DNA fragments to full chromosomes, and the production of devices on a very large scale for parallel processing; 4) because these structures can also be fabricated with extremely regular sized and spaced sieve features, the separation resolution should be improved leading to further miniaturization of the electrophoresis device, including the possibility of an electrophoresis analyzer on a chip; and 5) because the sieves can be built from inert substances analysis can be made of a variety of biomolecules.
One example of a micro-fabricated array formed using ion-beam lithography was disclosed by Duke et al., in Electrophoresis, vol. 18, pages 17-22 (1997), incorporated herein by reference. Duke et al. produced a periodic array of pillars 100 nm in diameter and 100 nm apart and demonstrated the ability to differentiate the electrophoretic mobility of DNA molecules between 7.2 and 43 kilobases (kb). Other disclosures of lithographically produced arrays for the use in electrophoresis devices are described in U.S. Pat. Nos. 5,110,339 and 5,837,115, both of which are incorporated herein by reference.
While this method clearly shows promise for providing nano-scale array sieves for separating DNA molecules having several thousand base pairs, the lower limit for array features made using such lithographic techniques is about 100 nm, indicating that smaller DNA molecules cannot be separated. Moreover, due to the time-intensive nature of e-beam lithography, it is not suitable for the economical fabrication of large dense arrays of with pore sizes approximately below 50 nm. This in turn limits the application of such sieves to DNA separation but not sequencing, which requires the separation of DNA molecules as small as a few base pairs long. In order to provide separation of molecules of up to 600 bases, inter-post separations of approximately 15 to 30 nm will be needed. Moreover, despite the progress made in recent years in developing new techniques to produce smaller and smaller features via lithographic techniques such as ion beam or electron beam lithography, it is clear that increasingly costly efforts are being required to sustain the progress. While deep UV and X-ray lithography offer hope of incremental improvements in resolution, lithographic and patterning techniques for reproducibly producing features at the 10 to 30 nm level are essentially nonexistent at the present time. The resolution limit of currently available patterning technologies make it apparent that entirely new approaches will be needed to sustain the rapid progress that has characterized the last few decades of semiconductor technology development.
One novel approach to making nanometer-scale structures utilizes self-assembly of atoms and molecules to build up functional structures. In self-assembled processing, atom positions are determined by fundamental physical constraints such as bond lengths and angles, as well as atom-to-atom interactions with other atoms in the vicinity of the site being occupied. Essentially, self-assembly uses the principles of synthetic chemistry and biology to xe2x80x9cgrowxe2x80x9d complex structures from a set of basic feedstocks. Utilizing such techniques molecular motors have been synthetically produced containing fewer than 80 atoms. Two relatively simple examples of self-assembled structures formed using chemical vapor deposition (a process commonly used in thin film deposition, including crystal-growth) include carbon fullerenes (xe2x80x9cbuckyballsxe2x80x9d) and carbon nanotubes. In addition, dense arrays of carbon nanotubes have been grown on surfaces and such arrays have been utilized as nano field emitter arrays, atomic force microscopy probes, nanoscale transistors, actuators, high-Q mechanical resonators and a variety of sensors.
While fabrication processes based on self-assembly at the nanometer scale have a number of advantages over the conventional lithography techniques, including avoiding the blanket film depositions, lithography, and subtractive processing characteristic of conventional nano-scale manufacturing, until recently there has been no method based on self-assembly for fabricating uniform nano-arrays of the type necessary to build an instrument for electrophoretic separation. Instead, the nano-arrays formed have been disordered on a fine scale with uncontrolled spacing between individual nanotubes and a significant spread in nanotube diameters.
Recently, a technique has been developed that relies on self-assembly to produce geometrically regular nanotube arrays with excellent uniformity. This process is based upon the self-organizing formation of highly uniform pore arrays in anodized aluminum films. First, a nanochannel alumina structure is formed by anodizing an aluminum film under conditions that lead to hexagonally-ordered arrays of narrow channels with very high aspect ratios. The nanochannel alumina structure is then used as a template for the growth of nanotube arrays of carbon and other materials, including metals and some semiconductors. The full process technique is disclosed in Appl. Phys. Lett., vol. 75, pg 367 (1999), and is incorporated herein by reference. Utilizing this technique the authors were able to grow nanotube arrays comprising uniform carbon nanotubes with a diameter of 32 nm. Despite the promise of this new technique, there have been no attempts to adapt the technology to grow array structure capable of being used as an electrophoretic sieve for analyzing and sequencing DNA and RNA.
Accordingly, a need exists for a durable non-gel sieving device capable of analyzing a wide range of molecular sizes under the influence of ultra-high electric fields and capable of being produced on a large scale for parallel processing.
The present invention is directed to a device and system for utilizing a non-gel self-assembled nano-array molecular sieve for analyzing biomolecules. In one particular embodiment this invention utilizes a non-gel ordered self-assembled array of nano-features that functions as an electrophoretic sieve to separate biomolecules. This invention is also directed to systems for integrating the non-gel ordered self-assembled nano-array sieve of this invention into a device for separating biomolecules. This invention is also directed to novel methods for separating a wide range of biomolecules using the non-gel ordered self-assembled nano-array sieve of the invention.
In one embodiment, the self-assembled nano-array of the present invention is incorporated into an electrophoretic micro-device comprising a self-assembled nano-array according to the present invention, a field generator for applying an electric field across the sieve, and a detector. The self-assembled nano-array sieve is designed to differentially transport polyelectrolytes, such as DNA molecules, proteins, etc., through the sieve in the presence of an electric field to produce a characteristic distribution of the introduced molecules. The nano-array is placed in line-of-sight with the detector such that as the molecules exit the sieve, they flow through the optical detection region of the detector. The detector then analyzes the exiting molecules such that the identity of the exiting molecule can be determined.
In one embodiment, the self-assembled nano-array sieve comprises a substrate having a periodic array of features such that in the presence of an appropriate feedstock the atoms of the feedstock self-assemble on the ordered features of the substrate to produce an ordered array of nano-features having non-random alignment and size distribution. In one embodiment the size, shape and pattern of the self-assembled nano-array features grown on the substrate are adapted such that molecules within a specified size range can be separated. The substrate wafer is preferably made of a substance that reacts with the feedstock to produce the self-assembled structures.
In another embodiment the nano-array features self-assemble into nanotubes having a specified diameter and height suitable for use in the sieve of the current invention.
In an alternative embodiment, the substrate is made of a semiconductor such as, for example, oxidized silicon or aluminum oxide, coated with a metal catalyst film such as, for example, Ni or Co. In this embodiment, the silicon can be further doped to adjust the electronic properties of the substrate surface.
In another alternative embodiment, the self-assembled nano-array sieve is confined in a channel and the outer surface of the array optionally covered with a cap layer so as to enclose the nano-array sieve such that the DNA molecules are confined within the sieve during separation. In such an embodiment, either the cap layer or the substrate must be transparent to light such that optical detection schemes can be utilized to analyze the molecular distribution. In this embodiment, reservoirs can be integrated into the channel and cap to provide an entrance point for the molecules to be tested.
In another alternative embodiment, the nano-array features are self-assembled from an inert material such as, for example, carbon utilizing a carbon feedstock gas such as, for example, acetylene.
In yet another alternative embodiment, the detector comprises a laser induced fluorescence system such as, for example, a laser diode emitter in conjunction with a binary superimposed grating, or conventional optics and a photomultiplier tube.
In still another alternative embodiment, the electrophoresis system of the present invention is utilized in combination with a DNA sequencer. In such an embodiment, the DNA strands would be labeled with a specific dye for each of the chain-terminating dideoxynucleotides and a binary superimposed grating in conjunction with four spatially separate optical detectors would be utilized to determine the identity of the tested molecules. In this embodiment, preferably the carbon nanotube array would have a pore size of about 15 nm and the molecules would have a size up to about 1,000 bases long.
In still yet another alternative embodiment, the invention is directed to a system for the detection of substances comprising multiple sieve paths and detectors as described above, such that parallel processing of molecules can be carried out.
In still yet another additional embodiment, the invention is directed to a method for separating molecules based on molecular size. The method comprises analyzing molecules introduced into the self-assembled nano-array sieve as described above.